Fabric for the preparation of electrophoresis gels

ABSTRACT

The invention relates to new fabrics with improved properties for the preparation of electrophoresis gels.

The invention relates to new fabrics with improved properties for thepreparation of electrophoresis gels and processes for producing them.

Polyacrylamide gels and agarose are used in numerous electrophoresistechniques as anticonvective and screening matrices. The gels are usedin two forms:

1) cylindrical gels, which were very popular for some time, are nowadaysparticularly preferred for first-dimension separation in two-dimensionalelectrophoresis; and

2) Flat gels, which are used in the techniques most frequently practisednowadays, namely sodium dodecylsulphate (SDS) electrophoresis andisoelectric focussing.

A fact common to both configurations is that during the separation thegels are supported from the outside, e.g. by the walls of a glass testtube or a sheet of glass. After separation the gels are evaluated, e.g.by protein staining, enzyme visualisation or blotting. The unsupportedgels are subjected to considerable mechanical stress, which means thatgels with a layer thickness of less than 0.7 mm cannot be used.

A further considerable disadvantage is that conventional gels are notdimensionally stable and will readily change their dimensions byswelling or shrinkage in solvents of different composition, making itdifficult or impossible to evaluate the components visualised.

By polymerising the gels onto suitably pretreated polyester films it ispossible to overcome to some extent the disadvantages described above.Using suitably pretreated polyester films it is possible to prepareultrathin gels, i.e. those with a thickness of about 50-350 microns,which have crucial practical advantages over gels of conventionalthickness (e.g. 1 to 3 mm) in isoelectric focussing and SDSelectrophoresis. However, film-supported gels of this kind have thedisadvantage that they are not optimal for capillary blotting and arecompletely unsuitable for electroblotting.

An alternative to the externally stabilised gels are fabric-supportedgels with internal stabilisation. Such gels are open on both sides andare therefore particularly suitable for blotting. However, they are alsoof interest for other applications, e.g. preparative isoelectricfocussing. Contrary to expectations, however, it has been found thatsuch electrophoresis gels prepared on fabrics do not meet the practicalrequirements.

Hitherto, it has been assumed that because of the greater contactsurface between the fabric and the gel, as compared with gels on supportfilms, the fabric-supported gels will adhere adequately even tonon-pretreated fabrics. This may be true for those applications in whichthe fabric-supported gels are merely stained after electrophoreticseparation, since no very great demands are made of the gel adhesion insuch cases. However, the situation is different in the application offabric-supported gels which is currently of most importance, namelyblotting. In this technique, after separation from the gel usingsuitable membranes, e.g. nitro-cellulose membranes, a blot is producedin which the separated components are immobilised so that they are thenavailable for further reactions, e.g. immunological detection methods.The transfer of the separated components into the membrane requires goodcontact between the surface of the gel and the nitrocellulose membranesince otherwise some of the resolution achieved in the gel will be lost.

The transfer may be carried out either by capillary blotting or byelectroblotting in an electrical field. In both transfer techniques,pressure is applied to the gel and the membrane in order to obtain asaccurate a copy as possible in the blot.

For further evaluation of the blot the membrane must be lifted away fromthe surface of the gel. It has been found, unexpectedly, that theadhesion of the gels to the supporting fabric is inadequate. Parts ofthe gel or, in some cases, the entire surface of the gel is or arepulled away from the membrane as well, so that subsequent evaluation ofthe blot is impaired or even rendered completely impossible.

A further disadvantage of the fabric-supported gels used hitherto isthat these gels frequently cannot be totally destained and consequentlythey have an uneven background. This may have a detrimental effect onfurther evaluation of the gels.

An object of this invention is therefore to provide industrial fabricswhich make it possible to produce fabric-supported electrophoresis gelswith improved properties, such as during evaluation.

We have found that the object may be achieved by using an industrialfabric which has a single or multilayer hydrophilic coating as anadhesion promoter.

Suitable fabrics are known from the prior art and are marketed byvarious companies. Industrial fabrics consisting of polyester with adefined mesh size of between 10 and 100 microns are preferred; fabricsof polypropylene or polyamide are also suitable. When choosing thefabric it should be ensured that it will not be damaged by theaggressive reagents or solvents frequently used in electrophoresistechnology from time to time. Fabric-supported gels are preferably usedfor the preparation of ultrathin electrophoresis gels (50 to 500microns), which means that the industrial fabrics used should have athickness of between 40 and 70 microns, preferably 50 to 60 microns.

Adhesion promoters according to the invention include those compoundswhich are able to bring about cross-linking between the fabric and thegel which is subsequently to be applied. Suitable compounds are forexample polymers and copolymers with reactive functional groups capableof crosslinking with the gel. German Offenlegungsschrift 30 32 069discloses suitable copolymers based on maleic anhydride derivatives withunsaturated primary or secondary amines or epoxides containing C═Cdouble bonds.

Copolymers of maleic anhydride and vinylalkylethers, copolymers ofpolysaccharides and allylglycidylethers and surfactants are alsosuitable.

Preferred adhesion promoters (crosslinking agents) include copolymers ofmaleic anhydride and vinyl-methylether (e.g. Gantrez AN obtainable fromServa Technik GmbH of Heidelberg, Germany) with diallylamine; agaroseand allylglycidylether (allylglycidyl agarose), or copolymers of maleicanhydride and methylvinylether (e.g. Gantrez AN), the preferredsurfactant being Surfynol.

The coating according to the invention may consist of a single layer ormay be made up of several layers. Multi-layer coatings may consist ofcombinations of individual layers which may contain different copolymersas adhesion promoters. If desired, a further coating of a surfactantsuch as 2,4,7,9-tetramethyl-5-decin-4,7-diol, may be applied to themono- or multi-layered coating of crosslinking agent. In anotherembodiment the surfactant may be added directly to the adhesion promoteras a further component.

The composition and structure of the coating are most suitably adaptedto the gel which is to be applied later.

The thickness of the coating is variable and may be adjusted, forexample, during preparation by adjusting the viscosity of the solutionsof adhesion promoter used during the immersion process. However, it isalways necessary for the weave-like structure of the industrial fabricto be retained, i.e. the meshes of the fabric should not be closed up bythe coating since otherwise the properties of the fabric supported gelwill be adversely affected.

Fabrics according to the invention suitable for the preparation of afabric supported polyacrylamide gel may, for example, have the followingstructure;,

a) a polyester fabric with a pore size of between 50 and 100 microns,preferably 60 microns, and a thickness of between 40 and 70 microns.

b) a 1st coating consisting of a copolymer of Gantrez and diallylamine,

c) a 2nd coating of allylglycidyl agarose, and

d) a 3rd coating of a surfactant e.g. Surfynol 104 (available from ServaTechnik GmbH, Heidelberg, Germany)

Another fabric by way of example according to the invention for thepreparation of a fabric supported agarose gel has a two-layer coating;e.g.

a) a polyester fabric with a pore size of between 50 and 100 microns,preferably 60 microns,

b) a 1st coating of Gantrez AN, and

c) a 2nd coating of agarose.

Since the fabrics used are generally sold for other purposes (e.g. asscreens), it may be advantageous to pretreat them in order to improvethe adhesion of the coating. This may be done, for example, simply bywashing them with water. The coatings may readily be preparedanalogously to known immersion methods.

For this purpose, the copolymers or surfactants used are dissolved insuitable solvents, if possible volatile solvents. Suitable solventsinclude, for example, acetone, methanol, water and others, whilst careshould be taken to ensure that the solvent does not damage the fabricsused. The fabrics are then dipped into the solution so that they arethoroughly wetted; the excess solution is then allowed to drip off andthe now coated fabric is carefully dried. The fabric is preferably driedat elevated temperature. Additional coatings of the same or a differentcomposition may subsequently be applied using the same process.

The fabrics thus prepared, once they have been carefully dried, may thenbe used to prepare fabric-supported electrophoresis gels. Thecorresponding techniques are known and need no further explanation.

The electrophoresis gels prepared from the fabrics according to theinvention have considerable advantages over those prepared fromconventional fabrics:

Fabric-supported gels prepared with fabric pretreated according to theinvention have significantly better adhesion, which can be determined,for example, in a test using a dry membrane. Moistened membranes such asare used in blotting can readily be detached from the surface of the gelwithout tearing away even part of the gel.

A further advantage of the pretreated fabrics becomes apparent duringmanufacture of the fabric-supported gels. The strongly hydrophobicproperties of the untreated fabric make it more difficult for thepolymerisation solution to penetrate during preparation of ultrathingels.

This disadvantage is substantially eliminated with the industrialfabrics treated according to the invention. The fabrics according to theinvention make it possible to prepare ultrathin electrophoresis gelswith thicknesses of between 50 and 500 microns, the preferred gelshaving a thickness of about 150 microns.

In addition to this advantage, the pretreated fabrics also show improvedproperties with regard to destaining and transfer efficiency. Whereasuntreated fabrics often have an uneven background during destaining, thetreated fabrics are practically uniformly destained. The transferefficiency is also better with the treated fabrics. This ensures thatthe proteins are quantitatively transferred from the fabric to themembrane.

The following non-limiting examples illustrate embodiments of theinvention:

EXAMPLE 1 Industrial Fabric for the Preparation of a Fabric SupportedPolyacrylamide Electrophoresis Gel

A fabric consisting of polyester (PES Monodur 60N, from Messrs.Verseidig, Kempen) is treated as follows.

a) Preparation of the first coating layer: The fabric is immersed in asolution* of 6 liters of acetone, 381 mg of Gantrez AN 179 (Serva^(R)),22 ml of DMF and 237.6 mg of diallylamine so that it is thoroughlywetted. Excess solution is then allowed to drip off and the fabric isdried at room temperature.

*The solution is only ready for use after 24 hours.

b) Preparation of the second coating layer: The fabric treated asdescribed in a) is dried and then immersed in a 0.2% solution ofallylglycidyl agarose in water and then dried.

c) Preparation of the third coating layer: After drying the fabric isfinally immersed in a 0.1% ethanolic solution of Surfynol 104(Serva^(R)) and dried.

The fabric thus prepared is then placed, in accordance with knownmethods, namely the so-called flap technique or cassette technique, inthe monomer mixture of acrylamide and conventional additives (such aspolymerisation initiator and other constituents, which then polymerisesto form the gel.

EXAMPLE 2 Industrial Fabric for the Preparation of a Fabric SupportedAgarose Electrophoresis Gel

The polyester fabric (PES Monodur 60N) is prepared by the immersionmethod as described in Example 1.

    ______________________________________                                        1st coating                                                                   Composition of   8 liters of acetone                                          the solution:    8 g of Gantrez                                               2nd coating                                                                   Composition of   8 g of agarose/5.6 ml water                                  the solution:    6 liters of methanol                                                          20 ml of glycerol                                            ______________________________________                                    

The embodiments of the invention in which an exlusive property orprivilege is claimed are defined as follows:
 1. A fabric useful for theproduction of electrophoresis gels comprising a polyester fabric havinga mesh size of between about 10 to about 100 μm, and also having amultilayer coating consisting of a first layer of a copolymer of maleicacid anhydride and vinylmethylether with diallylamine, a second layer ofallylglycidyl agarose and a third layer of a surfactant.
 2. A fabricuseful for the production of electrophoresis gels comprising a polyesterfabric having a mesh size of between about 10 to about 100 μm, and alsohaving a two-layer coating consisting of a first layer of a copolymer ofmaleic acid anhydride and vinylmethylether and a second layer ofagarose.
 3. A fabric useful for the production of electrophoresis gelscomprising a polyester fabric having a mesh size of between about 10 toabout 100 μm and also having a coating, in at least two layers, ofallylglycidyl agorose.
 4. The fabric as recited in claim 3 wherein theouter layer of coating also comprises a surfactant.
 5. Afabric-supported electrophoresis gel comprising a self-supportingpolyester fabric having a mesh size of between about 10 to about 100 μmand a thickness of between about 40 to about 70 μm, the polyester fabrichaving a multilayer coating consisting of a first layer of a copolymerof maleic acid anhydride and vinylmethylether with diallylmine, a secondlayer of allylglycidyl agarose and a third layer of a surfactant, and anelectrophoresis gel supported thereon.
 6. A fabric-supportedelectrophoresis gel comprising a self-supporting polyester fabric havinga mesh size of between about 10 to about 100 μm and a thickness ofbetween about 40 to about 70 μm, the polyester fabric having a two-layercoating consisting of a first layer of a copolymer of maleic acidanhydride and vinylmethylether and a second layer of agarose, and anelectrophoresis gel supported thereon.
 7. A fabric-supportedelectrophoresis gel comprising a self-supporting polyester fabric havinga mesh size of between about 10 to about 100 μm and a thickness ofbetween about 40 to about 70 μm, the polyester fabric having a coating,in at least two layers, of allylglycidyl agarose, and an electrophoresisgel supported thereon.
 8. The fabric-supported electrophoresis gel asrecited in claim 7 wherein the outer layer of coating also comprises asurfactant.
 9. The fabric-supported electrophoresis gel according toclaim 5, the electrophoresis gel being polyacrylamide.
 10. Thefabric-supported electrophoresis gel according to claim 6, theelectrophoresis gel being agarose.